Allogeneic Hematopoietic Cell Transplantation (HCT) is a widely used therapy for a variety of malignant and nonmalignant hematologic diseases. More than 8,500 patients, receive HCT transplants in the US each year. Yet, up to 50% of patients suffer complications due to graft-versus-host disease (GVHD) in the first year after HCT. GVHD occurs when donor immune cells attack the patient?s own tissues. GVHD can occur anywhere, with the skin, gut and liver most frequently affected. GVHD remains one of the major barriers to a more widespread application of allogeneic HCT. Early diagnosis of aGVHD is critical to inform treatment decisions and prevent serious organ injury and death. Unfortunately, few informative diagnostic approaches are available. In current clinical practice, diagnosis of aGVHD relies almost entirely on the presence of clinical symptoms and requires confirmation via invasive biopsy procedures, such as skin biopsy, colonoscopy, upper endoscopy or liver biopsy. HCT patients urgently need a better alternative. The goal of this proposal is to develop and apply a blood test to detect acute GVHD (aGVHD) and to predict long-term HCT outcomes. This test employs genome-wide profiling of methylation marks comprised within circulating cell-free DNA (cfDNA) in blood to trace their tissues-of-origin, and to quantify tissue-specific injury after HCT. This concept is supported by significant pilot data. We will perform a retrospective study of the utility of cfDNA to stratify HCT recipients with single organ aGVHD (Aim 1), and a prospective study of the utility of cfDNA to detect the early onset of GVHD and to predict HCT risk (Aim 2). Together these aims explore the highly innovative concept that a measurement of the tissues-of- origin of cfDNA in plasma enables to i) detect aGVHD, ii) quantify the severity of aGVHD and determine its organ involvement, iii) predict post-HCT mortality, and iv) predict the outcome of aGVHD treatments. This proposal directly addresses an urgent, unmet medical need: the early and noninvasive diagnosis of aGVHD after HCT. These studies are therefore highly translational. A highly informative, noninvasive monitoring tool may inform new treatment modalities and transplant strategies. Recognizing the limitations of universal prophylactic therapy against aGVHD, cfDNA assays could find future use in guiding pre-emptive treatment strategies, thereby reducing the need for prolonged immunosuppressive therapy after transplant. Furthermore, earlier detection of aGVHD could lead to shorter durations of therapeutic immunosuppression and improved patient outcomes.